Process for removing growth factors from platelets

ABSTRACT

In vitro and in vivo application of sub-atmospheric, negative pressure on growth factor starting material, such as whole blood, extracts growth factors from the platelet granules of the growth factor starting material in a non-destructive medium without activating the clotting process. The extracted growth factors are released into a growth factor composition containing blood plasma, extracellular fluid or interstitial fluid depending upon the type and location of the growth factor starting material. The growth factors have a weight of about 70-76 kDaltons and are applied in either a filtered or unfiltered state topically to the area of a surface wound to effect healing. The extracted growth factors are also injected into soft tissue, such as a torn tendon, to promote tissue growth and healing. The growth factors are released in one method from a patient&#39;s own blood. In another method the growth factors are released from a whole blood source and freeze dried by lyophilization. Then at a later date, the freeze-dried product is reconstituted by normal saline for treatment of a patient&#39;s wound, for use in a surgical procedure, or for tissue regeneration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.14/120,487 filed on May 23, 2014 which is a division of U.S. applicationSer. No. 12/459,911 filed Jul. 9, 2009, now U.S. Pat. No. 8,734,854.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a process for extracting growth factors fromgrowth factor starting material and other cellular components fromplatelets, platelet rich plasma, and whole blood and, more particularly,to a wound healing and tissue regenerative composition of growth factorsand cellular components released from intracellular granules andcellular structures bound by mammalian platelet membranes for use inwound healing and other therapeutic and biomedical uses to repair,regenerate, restore and provide bio-scaffold components to promote andsupport living cells and tissues and for the purpose of stabilizing orreversing impairment of the normal state of the living animal body orone of its parts whose function has been interrupted or performance ofthe vital functions compromised by injury, disease, or aging.

2. Description of the Prior Art

The practice of using activated autologous platelets as a treatment in anumber of medical and surgical procedures is known, including but notlimited to oral and maxillofacial surgery, orthopedic surgery, cosmeticand reconstructive surgery, chronic tissue repair, sports medicineinjuries, neurosurgery, cardiovascular surgery, podiatry, hairtransplant surgery, immune mediated hair loss, medical research, tissueengineering, and non-surgical cellular therapy. U.S. Pat. Nos. 4,957,742and 6,649,072 disclose wound healing compositions that include plateletrich plasma (PRP) which prior to use is activated by thrombin to releasegrowth factors from the alpha granules of the platelets.

Extracting therapeutic levels of platelets has been a technicalchallenge requiring trained cardiovascular perfusionists to operate theequipment originally designed for the production of platelet richplasma. The clinical practitioner now has access to more simplifiedequipment that allow processing of PRP with smaller amounts of wholeblood in a shorter amount of time. Venous access, clinical expertise,and cost are still challenges that have limited the widespread use ofthis process throughout the world. Moreover from a commercialstandpoint, wound-healing compositions that include platelets must meetcostly FDA guidelines applicable to blood products.

Platelet rich plasma (PRP) compositions are being utilized for bothhuman and veterinary applications. PRP formulations may or may notinclude leucocytes (white blood cells) and are referred toleucocyte-rich PRP or leucocyte-poor PRP. As the knowledge of growthfactors expands, a greater understanding of specific growth factors hashelped to define their roles with greater precision. In general, whileplatelets influence anabolic signaling to promote the proliferative andregenerative phases of the healing cascade, leucocytes containcytokines, a class of growth factors with catabolic activity supportiveof an inflammatory response. Thus, a PRP preparation or a growth factorcomposition can be tailored to the desired anabolic or catabolicactivities through selective inclusion or exclusion of leucocytes.

Growth factors are responsible for the wound healing process, asdescribed above. Platelets function as carriers for the growth factors.Growth factors are polypeptides produced by the tissue on which theyact. They regulate differentiation, proliferation, migration, andmetabolism in target cells, regulating the synthesis of specificadhesion molecules that control cell-cell and cell-substrateinteractions. Each GF can have either one or several essential functionsfor a specific cell, depending on the particular circumstances of thecell environment.

The most widely studied growth factors in relation to tissueproliferation and repair include: bone morphogenetic proteins (BMPs)(eg, BMP-1, BMP-2, and BMP-3); PDGF; insulin-like growth factor (IGFs)(eg, IGF-I and IGF-II); TGFs, especially TGF-8; fibroblast growthfactors (FGFs) (ie, acid-FGF and basic-FGF); granulocyte macrophagecolony stimulating factor; epidermal growth factor; and VEGF. Animportant role in repair processes, specifically in the inflammationstages, is also played by cytokines produced by white blood cells,including the interleukins (ILs) IL-1, IL-3, IL-6, and IL-8. All ofthese growth factors and cytokines act to a greater or lesser extentduring the different stages of wound healing which includes tissuenecrosis resolution, cell regeneration, cell proliferation andmigration, extracellular matrix synthesis, epithelialization, andremodeling.

There is need for an efficient process for extracting and isolatinggrowth factors and platelet granule contents from the plateletscontained in plasma for subsequent use in wound healing and for amultitude of bioactive processes supportive of living tissue(s)associated with in vivo or in vitro processes. Preferably, the finalproduct would be a growth factor composition with minimal to no cellulardebris.

It is further desirable to prepare a wound healing product, expanded toinclude processes as described above, that can be subjected toconventional preservation procedures, such as lyophilization, freezedrying, and cryopreservation in a process that does not destroy thegrowth factors nor the functionality of the growth factor composition.In this manner the shelf life of the product(s) would be significantlyprolonged.

In addition to local hemostasis at sites of vascular injury, plateletscontain an abundance of growth factors and cytokines that are pivotal insoft tissue healing and bone mineralization. An increased awareness ofplatelets and their role in the healing process has led to the conceptof therapeutic applications.

The preparation of PRP is still dependent on whole platelets,centrifugation or gravity flow or filtration techniques with theexception of cytokine rich plasma (CRP), as discussed in U.S. Pat. No.8,734,854. During the early years of development, the terminologycytokine rich plasma represented both growth factors and cytokines. Morerecently growth factors have come to represent positive tissue repairinfluences and regeneration while cytokines are associated withinflammation, pain, and cleaning up of wounds.

It should be understood that CRP composes both classification types butmay be better thought of as complex or complete rich plasma. CRP isunique to PRP in both composition and concentration. Although similar inmany ways, the differences are relevant. Intact platelets are common toall forms of PRP. CRP is developed under the influence ofsub-atmospheric pressures. Growth factors, cytokines and otherintracellular proteins and biochemical components normally released byagonists are extracted from their intracellular positions without theuse of agonists. The result: CRP is overwhelmingly acellular.

Production of PRP inherently results in less than 100% of the originalcontent of whole blood cell types of interest. Production of CRP may bederived from 100% of the cellular content of whole blood. Whole blood orvarious blood cell components, specifically platelets and or white bloodcells, are subjected to sub-atmospheric pressures. Maximal levels ofeach sample's cellular proteins are separated from their intracellularlocation and released to the extracellular (extracorporeal) fluidenvironment. Additionally, assays of the sub-atmospheric treated bloodled to findings of two peptides not previously recognized and isdisclosed in U.S. Pat. No. 8,734,854.

Processing the blood components after being exposed to controlledsub-atmospheric pressure allows for separation of CRP from red bloodcells, ghost cells and cell debris resulting in an acellular content.The CRP can then be directly applied to a patient; filtered to removewater thereby concentrating the product into a smaller volume forapplication in areas where smaller volumes are more efficacious, such aswithin the confined space of joint, or stored by a unique lyophilizationprocess. Discovered by accident, this specific method of lyophilizationpreserves biofunctionality, the ability to express bioactive properties,of the CRP components without the addition of fixatives. The latterprocess remains a trade secret to the inventors.

It has long been the goal to finding a means of preserving or extendingthe functional longevity of the platelet. Utilizing the concept ofnegative (sub-atmospheric) pressure, living tissues composed of wholeblood and portions thereof were subjected to controlled levels ofnegative pressures. Under the appropriate range of negative pressure,the blood cell maintains its integrity while undergoing expansion. Toomuch negative pressure and the cell tears apart. Not enough negativepressure and the cell will not react. With the appropriate degree ofnegative pressure applied over a defined timeline, the cell expandsreducing internal pressures such that particulate matter contained withintracellular alpha, delta (dense) or lambda granules find a pathway ofleast resistance as they are freed from their confines of the expandinggranule membrane. These intra-granule particles are drawn to the lowerpressure outside the cell membrane and into the extracellularenvironment. The contents of the granules are emptied without destroyingthe integrity of the cell membranes.

The natural life expectancy of a platelet and its granular contents arelimited to days using standard blood banking processes. Platelets arecytoplasmic fragments of megakaryocytes lacking nuclei but containingorganelles and structures such as mitochondria, microtubules, and threemain types of granules. Due to ongoing mitochondrial activity, and mRNAsynthesis of proteins, this meets the description of a living cell.These′short-lived cells or cell fragments, whichever one prefers to viewthem as, have undergone extensive research in order to come up with areliable means of preservation with retention of normal bioactivepotential. For this purpose, platelets will be viewed as cells.

Platelets reside intravascularly. Therefore, any tissue containing avascular supply will have the same platelet content. The normalconcentration of platelets in blood is approximately 140,000 to 400,000platelets/mm3. These remain in the circulation for about 10 days.Therefore any disruption in one's ability to replace platelets wouldhave rapid and profound effects in the event of a sustained trauma.After tissue injury, platelets are among the first cells to appear andremain in the vicinity of the wound.

The benefits of controlling the collection of and when desirable, thestorage of this limited resource, to extend the useful function of aplatelet or its contents, would have significant impact on the practiceof medicine as it applies to wound healing, regenerative medicine, andother therapeutic and biomedical uses for the purpose of stabilizing orreversing impairment of the normal state of the living animal body orone of its parts whose function has been interrupted or performance ofthe vital functions compromised by injury, disease, or aging.

Historically, the main purpose of preserving the platelet pertains tocorrecting bleeding disorders stemming from deficiencies in plateletnumbers or platelet function. Advancing technologies have allowed for agrowing body of evidence to reveal the critical and diverse role theplatelet in the wound healing cascade that includes the steps ofhemostasis (clotting), inflammation, proliferation, and regeneration(remodeling) of tissues. Platelets regulate and modulate the rate oftissue repair by releasing biochemical messages, growth factors, thatcan affect the cell from which it originates (autocrine), or influencelocal cell activity (paracrine) or distant (endocrine) cell activity.This call to action influences multi-cell activity as well as themigration of healing components to the site of injury.

Over 1200 types of proteins have been identified on or within aplatelet. There are approximately 50 to 80 alpha granules per plateletcontaining greater than 400 different bioactive proteins whose complexinteractions in the healing cascade are not yet fully clarified. Densebodies or Delta granules (250-300 nm) contain ATP, proaggregatoryfactors such as adenosine 5′-diphosphate (ADP), calcium, and5-hydroxytryptamine (serotonin), pyrophosphate, histamine and otherfactors which promote adhesion of platelets and cause vasoconstriction.Lambda granules (175-250 nm) contain lysosomal, proteases, lipases,nucleases and polysaccharidases. These bioactive enzymes function toremove infectious agents and cellular debris.

It should be understood that PRP is more than just platelets and that itcontains many bioactive factors that act in anabolic, catabolic,proinflammatory, and anti-inflammatory pathways. Some components arealso involved in the modulation of the immune response. The precisecombination and concentration of platelets, leukocytes, and other plasmacomponents best for musculoskeletal healing are not presently known, andclinicians should be aware that the effects of PRP are not solely basedon platelet concentration. A maximal efficacious concentration beyondwhich the platelet concentration will provide no further clinicalbenefits likely exists. Although the effects of many of the proteins inPRP on musculoskeletal tissues are still unknown, they likely contributeto the biologic healing process. Finally, it is imperative forindividuals involved in clinical study design and all clinicians to takeinto consideration diurnal variation in platelet count and that, simply,generation of PRP will fail in some patients in some instances.

Activation of a platelet by an agonist, such as thrombin, collagen, orother agonist known in the art, leads to the differential release ofgranule material from within the platelet. Such granulation activation(differential degranulation) results in the specific and sequentialrelease of groups of growth factors and other granular contents overtime.

Physical agonists such as rapid cooling and freezing temperatures asused in standard lyophilization, cryopreservation, and freeze dryingprocesses result in a percentage of platelet destruction and asignificant loss of bioactivity rendering them less potent. Theappropriate application of sub-atmospheric pressure results in a rapidand thorough release of granular contents with retention of bioactivity.

The platelet cytoplasm contains two distinct pathways: first a closeddense tubular system which does not open to the cell membrane and secondan open cannicular system which appears to be an invagination of theouter cell membrane and does open to the cell membrane and through whichplatelet granular contents are dispersed when activated. Where the densetubular system lies adjacent to the open cannicular system, transfer ofproteins across membranes will occur. During the unconventionalapplication of a sub-atmospheric pressure without the use of natural orknown platelet activators, findings support that negative pressuresassist in the nonspecific extraction (release) and discharge of granularcontents into the dense tubular system and or cannicular system of theplatelet from which growth factors are released beyond the cell membraneto the extracellular (extracorporeal) environment. In effect, a rapidand complete, non-differential or nonspecific release of granularmaterial results from a sufficient, application of sub-atmosphericpressure.

Membranous pseudopod formation is discussed at length in the medicalliterature. Natural or agonist induced activated platelets undergopseudopod formation. Studies are lacking as to whether or notsub-atmospheric pressure will induce pseudopod formation but it ishypothesized that it will not occur with rapidly applied negativepressure. Rather the entire platelet cell will expand more uniformly.

Human platelet granules degranulate via the intracellular caninicularsystem and dense tubular system. Equine and bovine platelet granuleshave been shown to release growth factors directly from the cellularmembrane. Canine platelet degranulation appears to vary between cellmembrane and cannicular system. Various forms of disease have beenstudied that have an affect on the platelet degranulation process.

When applied to existing wounds, growth factors are known to attractmore platelets (proaggregatory factors), macrophages, regenerative cellssuch as mesenchymal stem cells and osteoblasts, and increase the rate ofcollagen laydown, vascular ingrowth, fibroblast proliferation andoverall healing. The release of a protein known as platelet-derivedgrowth factor (PDGF) serves as a chemotactic signal for monocytes,neutrophils and fibroblasts, which then move into the wound to begin theinflammatory stage of the healing process. During this time, monocytessecrete a number of factors, including PDGF and transforming growthfactor-beta 1 (TGF-β1) also found in platelets. In this mannerfibroblasts are activated to begin the repair or regenerative stage ofthe healing process. Subsequently, wound healing continues through theprocess of collagen remodeling within the wound.

Platelet rich plasma compositions are being utilized for both human andveterinary applications. PRP formulations may or may not includeleucocytes (white blood cells) and are referred to leucocyte-rich PRP orleucocyte-poor PRP. As the knowledge of growth factors expands, agreater understanding of specific growth factors has helped to definetheir roles with greater precision. In general, while plateletsinfluence anabolic signaling to promote the proliferative andregenerative phases of the healing cascade, leucocytes containcytokines, a class of growth factors with catabolic activity supportiveof an inflammatory response to help resist infection and remove cellularand tissue debris. Thus, a PRP preparation or a growth factorcomposition can be tailored to the desired anabolic or catabolicactivities through selective inclusion or exclusion of leucocytes.

Growth factors are responsible for the wound healing process, asdescribed above. Platelets function as carriers for the growth factors.This further understanding is clearly recognized and included with useof the terminology “growth factor composition” which is understood toinclude the contents of platelet alpha, dense, and lambda granules thatcontain over 400 different bioactive proteins and biochemicals whosecomplex interactions in the healing process are not yet fully clarifiedas well as components of the extracellular fluid or plasma. Therefore,there is a desire for an efficient process for extracting and isolatinggrowth factors and when appropriate, additional bio-substances, from theplatelets and the blood, plasma, or tissue(s) containing the plateletsat the time of application of the sub-atmospheric pressure forsubsequent use in wound healing and for a multitude of bioactiveprocesses supportive of living tissue associated with in vivo or invitro processes. The use of a natural growth factor composition as partof a supportive medium or bioscaffold to produce whole organs or tissuesin vitro has significant implications. Preferably, the final productshall be free of other selective components that are typically found inconventional platelet enriched wound healing products, namely wholeplatelets, ghost platelets, white blood cells, red blood cells,bacteria, and other cellular debris.

There is further benefit to preparing naturally derived wound healingproducts that can be subjected to conventional preservations, such aslyophilization, freeze drying, and cryopreservation in a process thatdoes not destroy the growth factors nor the functionality of the growthfactor composition. In this manner the shelf life of the product(s)would be significantly prolonged.

Therefore, there is a need for a process for isolating and extractinggrowth factors in a non-destructive manner from platelets. The resultingcomposition may selectively contain other platelet and if desiredplatelet rich plasma components or, may or may not be substantially freeof other components, such as whole platelets, ghost platelets, whiteblood cells, red blood cells and bacteria, and can be used fresh forimmediate use or lyophilized for delayed use into a shelf-stable,non-refrigerated product for subsequent use.

SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a process forobtaining growth factors that includes the steps of providing apreselected volume of unfrozen growth factor starting material whereinthe unfrozen growth factor starting material contains platelets obtainedfrom the tissue of a subject. A preselected sub-atmospheric pressure isapplied to the unfrozen growth factor starting material to extractgrowth factors from the platelet granules of the unfrozen growth factorstarting material without activating the clotting process within thegrowth factor starting material. The extracted growth factors arereleased from the cellular structure of the growth factor startingmaterial into extracellular fluid to provide an extracellular growthfactor composition containing the extracted growth factors. Theextracted growth factors are collected for mixture with a nondestructivemedium without dehydrating the growth factor starting material. Thenondestructive medium contains a therapeutically effective amount ofgrowth factors for creating a positive reaction on living tissue.

Further, in accordance with the present invention there is provided awound healing and tissue regenerative composition that includes unfrozengrowth factor starting material including platelet granules obtainedfrom the tissue of a subject. The growth factors are extracted from theplatelet granules of the unfrozen growth factor starting material. Theextracted growth factors remain in an inactivated state. The extractedgrowth factors are substantially free of cellular structure of thegrowth factor starting material. The extracted growth factors are mixedwith a nondestructive medium in a bioactive state to generate a positivereaction on living tissue to enhance tissue repair and or growth.

Additionally, the present invention is directed to a process forobtaining growth factors that includes the steps of providing apreselected in vivo site of growth factor starting material containingplatelets obtained from the tissue of a subject. A preselectedsub-atmospheric pressure is applied to the growth factor startingmaterial to extract growth factors from the platelet granules of thegrowth factor starting material over a preselected period of timewithout activating the clotting process within the growth factorstarting material. The concentration of available platelet free growthfactors is raised within the growth factor starting material to amagnitude of extracellular physiological concentration exceeding themagnitude normally found in extracellular physiological concentration.The extracellular physiological concentration of platelet free growthfactors upon application promotes an acceleration of the healingcascade.

Further, the present invention is directed to a process for producing atherapeutically and regeneratively effective biofunctional agent thatincludes the steps of extracting growth factors from platelets of growthfactor starting material derived from the tissue of a subject withoutactivating the growth factors and with the growth factors remaining in anatural state. A growth factor composition is produced including growthfactors extracted from platelets. The growth factor composition isseparated from the growth factor starting material. The growth factorcomposition is collected and applied as a catalyst in the formation ofan extracellular tissue matrix for creating a positive reaction onliving tissue.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of a resting platelet circulating in bloodplasma prior to activation by an agonist.

FIG. 2 is a schematic diagram of a platelet activated by an agonist,illustrating release of growth factors through the intracellular canalsystem of the platelet to the extracellular space of the plasma.

FIG. 3 is a schematic diagram of a platelet exposed to sub-atmosphericpressure, illustrating release of growth factors in the absence of anagonist through the intracellular canal system to the extracellularspace of the plasma.

FIG. 4 is a diagrammatic flow chart of the process for releasing growthfactors from platelets, illustrating the step of applying a hardcentrifugal spin to the growth factors after release from platelets.

FIG. 5 is a diagrammatic flow chart similar to FIG. 4, illustrating theapplication of a soft centrifugal spin followed by a hard centrifugalspin to the released growth factors.

FIG. 6 is a diagrammatic flow chart similar to FIGS. 4 and 5,illustrating the step of lyophilizing the growth factors after beingreleased from the platelets and subjected to a hard centrifugal spin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The process of the present invention removes growth factors fromplatelets for subsequent use in wound healing and tissue regenerativeprocesses, either alone or in combination with other healing componentspresent in plasma or other growth factor (cytokine) bearing cells suchas leucocytes. In accordance with the prior art processes, plateletconcentrated plasma products are prepared through multi-step processesand then subsequently activated with one or more known activationprocesses, such as thrombin or collagen, to release the growth factorsand other platelet granule components from the platelets' alphagranules. In contrast, the process of the present invention allows forthe separation of growth factors from normal and concentrated levels ofplatelets without the need to use activators (agonists), such asthrombin or collagen. Consequently, with the present invention plateletdegranulation and growth factor release is carried out without theoccurrence of clotting. As a result, a higher yield of growth factors isobtained. Further, adhesion molecules typically consumed in formation ofa clot are harvested for later use. The released growth factors remaininactivated so that they retain bioactivity and bio-messenger capacityseparate from or in the presence of fibrinogen, fibronectin, andvitronectin in a nondestructive medium, such as plasma, sterile water,saline, and the like. The bio-messenger capacity of the released growthfactors results in signaling a change or response in the behavior of aliving cell, including the cell of origin. In a bioactive state thereleased growth factors have a positive reaction on living tissue, whichin one example provides enhanced wound healing. The growth factors andadhesion molecules may be lyophilized, for example, to prepare afreeze-dried product with a shelf life much longer than non-lyophilizedplatelet products.

As used herein, the term growth factors refers to any material ormaterials having a positive reaction on living tissues, such aspromoting the growth of tissues. Exemplary growth factors include, butare not limited to, platelet-derived epidermal growth factor (PDEGF),platelet factor 4 (PF 4), transforming growth factor beta (TGF-β),acidic fibroblast growth factor (FGF-A), basic fibroblast growth factor(FGF-B), transforming growth factor A (TGF A), insulin-like growthfactors 1 and 2 (IGF-1 and IGF-2), B thromboglobulin-related proteins(BTG), thrombospondin (TSP), fibronectin, von Willibrand's factor (vWF),fibropeptide A, fibrinogen, albumin, plasminogen activator inhibitor 1(PAI-1), osteonectin, regulated upon activation normal T cell expressedand presumably secreted (RANTES), gro-A, vitronectin, fibrin D-dimer,factor V, antithrombin III, immunoglobulin-G (IgG), immunoglobulin-M(IgM), immunoglobulin A (IgA), a2-macroglobulin, aniogenin, Fg-D,elastase, keratinocyte growth factor (KGF), epidermal growth factor(EGF), tumor necrosis factor (TNF), fibroblast growth factor (FGF) andinterleukin-1 (IL-1), Keratinocyte Growth Factor-2 (KGF-2) andcombinations thereof. One of the important characteristics common to theabove listed growth factors is that each substance is known as orbelieved to have a positive reaction on living tissue, known asbioactivity, (anabolic) to enhance cell or tissue growth.

In the context of the healing cascade having positive effects on livingtissues, the inflammatory stage has distinct aspects associated with itin which tissues are prepared for a healthy recovery but in order toachieve this they must first go through a catabolic or clean up phase toeliminate devitalized cells and microbial and foreign body contaminants.Pain is associated with inflammation. Cytokines help to regulate thepain response. The presence and or absence of specific interleukinsdetermine the degree of pain experienced during the inflammatory phaseof healing. Chronic pain such as that experienced in osteoarthritisoccurs from prolonged cellular trauma where wear and tear on theaffected tissues never allow for complete healing. Therapies utilizingPRP, platelet and leucocyte derived interleukins have been recognized.This remains one exception to positive aspects of the healing cascadeand provides a platform for the use of growth factor compositions toovercome the pain response associated with chronic osteoarthritis.

Growth factor starting material as used herein refers to any source ofunfrozen animal tissue containing one or more growth factor components,such as platelets, PRP, whole blood, bone marrow, umbilical cord fluid,adipose tissue, vascular tissue, and combinations thereof. All embryonicderived tissues of mesodermal, endodermal and ectodermal origin aresources of growth factor starting material. For example, adipose tissueis composed of fat cells called adipocytes and vascular components. Thevascular components are composed of blood vessels containing cells withgrowth factor components. Therefore, adipose tissue is a source ofgrowth factors. Any tissue or organ with a blood supply contains growthfactor starting material.

FIG. 1 schematically illustrates an inactivated platelet or restingplatelet generally designated by the numeral 10. The platelet 10contains alpha granules 12 that are filled with growth factors 14 thatare released from the alpha granules 12 and platelet 10 in accordancewith the present invention. The platelet circulates in the blood plasmaready to respond if activated by one or more agonists. A membrane 16surrounds the platelet and includes receptors 18 positioned at themembrane 16. The platelet 10 includes an intracellular system within themembrane 16 and an extracellular system external of the membrane 16. Theextracellular system outside the platelet includes a fluid in which theplatelet circulates. The intracellular system includes a dense tubularsystem (not shown) and a cannicular system 20. The dense tubular systemis not open to the environment external of the platelet; however, asshown in FIG. 1, the cannicular system 20 is open to the environmentexternal to the platelet 10.

Referring to FIG. 2, there is schematically illustrated a platelet 10when activated by an agonist 22 at the site of a cell membrane receptor18. In the presence of an agonist the growth factors 14 are releasedfrom the alpha granules and may pass either through the internal densetubular system into the open cannicular system 20 or pass directly intothe open cannicular system 20. From the cannicular system 20, thereleased growth factors pass through openings in the platelet membrane16 into the extracellular fluid surrounding the platelet. As furtherillustrated in FIG. 2, not all of the growth factors are released fromthe alpha granules in the presence of an activist 22. A number of alphagranules remain intact within the platelet to be released at a latertime or not at all.

For improved clinical use of growth factors and to provide a diversifiedapplication of the growth factor composition of the present invention,it is important that the growth factor starting material not be frozenprior to separation of the growth factors from platelets. Preferably,the process should be performed above freezing temperatures, such asroom temperature. In the preferred embodiment of the present invention,platelet rich plasma (PRP) is employed as the source of growth factorsand may be obtained via methods known in the art.

Exemplary platelet plasma products are disclosed in U.S. Pat. Nos.6,214,338; 6,010,627; 5,165,928; 6,303,112; and 6,649,072. The moreconcentrated the plasma is with platelets, the greater the concentrationof growth factors that can be obtained via the present invention. Theprocess for isolating growth factors from platelet rich plasma or othermedia containing platelet rich plasma is described hereinafter ingreater detail.

As used herein, therapeutically effective amount of wound healingcomposition refers to the amount of the constituent bioactive elementsor combinations thereof necessary to form a wound healing composition.The wound healing composition forms an extracellular matrix,proliferation of granulation tissue, facilitation of collagen laydown,vascular ingrowth, fibroblast proliferation, and stem cell activation,producing a reduction in the volume or surface area of a wound. Allembodiments of the present invention are assumed to have minimal orgreater therapeutically effective amount(s) of constituent substances orcombinations thereof to possess the above positive bioactive properties.

Once the platelet rich plasma is obtained, it is placed under a vacuum,preferably under a sub-atmospheric or negative pressure. The PRP is inan unfrozen state, preferably at room temperature. Similarly, the vacuumis applied at above freezing and preferably at room temperatureconditions. Optionally, whole blood or portions thereof, can besubjected to a controlled sub-atmospheric pressure under like conditionsas described for PRP.

Sub-atmospheric pressure release can be artificially induced andcontrolled using devices with properties as described herein, whichsubject the platelet or growth factor starting material to a timeoriented, specified range of sub-atmospheric pressure. Following themethod described herein, the growth factor composition can be used onsite or within a few days if kept cooled or further processed bylyophilization, referenced earlier, to store long term. The resultingcomposition of growth factors and bio-components obtained through thislatter process retain normal bioactivity potential in a quiescent stateuntil applied to target cells or tissues. Similarly, applying acontrolled degree of sub-atmospheric pressure over a controlled periodof time “in situ” will initiate the extraction/release of growth factorsfrom platelets in the tissue(s) under the influence of negative pressureand results in a greater concentration of growth factor activity on theaffected tissue(s).

In accordance with the present invention, the PRP is placed in one ormore sterile vials and the vials placed in a vacuum chamber. A vacuum isapplied using a conventional vacuum pump preferably at temperaturesabove freezing. In one example, it is applied between 1° C. and 37° C.and at a sub-atmospheric, negative pressure preferably between 5millibars to 1 atmosphere. As a result of the application of thenegative pressure, the growth factors are released into the surroundingnondestructive medium contained within the vials. Optionally, manual orelectric driven devices can be substituted for the vacuum pump thatgenerates a controlled, sustainable negative pressure on the blood ortissue sample for a defined period of time. An example device is asealable container with a movable wall to confine the blood sample. Themovable wall effectively allows for the chamber in the sealed containerto expand without allowing for any exchange of air between the insideand outside of the container.

One device suitable for use for subjecting a patient's own growth factorstarting material to sub-atmospheric pressure is disclosed in U.S. Pat.No. 8,871,745. This manually driven apparatus processes a known quantityof autologous growth factor starting material and subjects the materialto a predetermined range of sub-atmospheric pressure. A built-in lockingmechanism maintains a consistent pressure over a selected period oftime. A growth factor composition is produced and extracted through apropositioned withdrawal port. The growth factor composition is anextracellular fluid or plasma containing the released growth factors,such as the contents of platelet alpha, dense, and lambda granules thatcontain over 400 different bioactive proteins and biochemicals whosecomplex interactions in the healing process are not yet fully clarified,as well as components of the extracellular fluid or plasma. Theextracellular fluid includes all body fluids outside of the cells,having two major components, interstitial fluid and blood plasma.Interstitial fluid is a solution that bathes and surrounds the cells ofmulticellular animals.

There is a direct inverted correlation of time versus negative pressurewithin the specified range of sub-atmospheric pressure. The shorter thelength of time the negative pressure is applied, the greater thenegative pressure must be. Conversely, the longer the length of time thenegative pressure is applied, the less the negative pressure needs to beto release the growth factors from the platelets. A vacuum pressuresource suitable for use in the process of the present invention is arotary vane direct drive vacuum pump commercially available fromLabconco Corporation of Kansas City, Mo. It should be understood thatother commercially available vacuum generating devices are operable foruse with the present invention.

As a result of the vacuum process, the growth factors are separated orreleased from the platelets in the growth factor starting material intothe extracellular fluid containing plasma and water, leaving theplatelets intact.

Now referring to FIG. 3, there is schematically illustrated the platelet10 when exposed to sub-atmospheric pressure in accordance with thepresent invention. The sub-atmospheric pressure when applied to theplatelet reduces the surface tension on the platelet membrane resultingin an increase in the opening of the cannicular system 20 through theplatelet membrane 16. The sub-atmospheric pressure also creates anexpansion of the dense tubular system resulting in expansion of thealpha granules and subsequent release of the growth factors. The growthfactors released from the expanded granules pass from the dense tubularsystem into the open cannicular system 20.

As further illustrated in FIG. 3, in response to the sub-atmosphericpressure the alpha granules 12 migrate to the open cannicular system 20so that the growth factors are removed from the alpha granules and passinto the cannicular system 20. Substantially all of the alpha granulesare affected by the sub-atmospheric pressure to the extent that they arereleased from all of the alpha granules in the platelet and pass fromthe intracellular cannicular system 20 to the extracellular environmentoutside of the platelet. The movement of the extracted growth factorsinto the extracellular fluid (plasma) surrounding the platelet forms thegrowth factor composition, which is ready for wound healing andtherapeutic application.

The plasma is a nondestructive medium, forming the growth factorcomposition. Plasma is the pale yellow liquid component of blood thatnormally holds the blood cells in whole blood in suspension. It makes upabout 55% of the body's total blood volume. It is the intravascularfluid part of extracellular fluid and contains mostly water (95% byvolume) and dissolved proteins (6-8% by volume). The growth factorcomposition can be concentrated by removing the water therein by ahemoconcentrating filter.

As shown in FIG. 3, the negative pressure created by the vacuuminitiates movement of the growth factors out of the platelet granulessurrounded by the platelet membrane and into the extracellular plasma.The extracted growth factors are collected for mixture with thisnon-destructive medium without dehydrating the growth factor startingmaterial. The non-destructive medium forms with the growth factorstarting material and the growth factor composition for application towound healing or tissue regeneration. Ghost platelets may also remainfor therapeutic use.

In one example of the process of the present invention, analysis of thevacuumed plasma using light microscopy and alpha granule stainingtechniques revealed intact platelets devoid of alpha granules (ghostplatelets) in addition to the presence of platelet derived growthfactors (PDGF) distributed in the plasma, as determined by lab assay.Platelet derived growth factors typically have a weight of 16-20kDaltons. Specific growth factors extracted from the platelets inaccordance with the present invention were measured to have an increasedweight of 70-76 kDaltons. This is likely to be the result of a limitedactivation of growth factors allowing for the formation of a stable,larger protein moiety.

In another example, the process of the present invention for isolatinggrowth factors is conducted using a composition of platelet rich plasma(PRP) and platelet poor plasma (PPP), as described in U.S. Pat. No.6,649,072 (“the '072 patent”). The composition disclosed in the '072patent is about a 3:1 ratio of PRP to PPP. With this ration of PRP toPPP, the amount of growth factors obtained includes platelet derivedgrowth factor-AB/BB 356673.86 pg/ml, vascular endothelial growth factor6440.667 pg/ml and platelet-derived epidermal growth factor 1106.73pg/ml. This constitutes a significant recovery of growth factors notfound in platelets that are released by the natural process of plateletactivation and not otherwise attainable for positive clinical use inapplying topically to a wound or injecting into soft tissue.

In another embodiment of the present invention, the growth factors arepreserved for future bioactive use by preservation methods, such aslyophilization, cryopreservation, and flash drying. In this manner, ashelf-stable product is produced that is usable for years afterpreparation when stored at room temperature. When desired for use, thelyophilized product is reconstituted with sterile 0.9% normal salinesolution. The lyophilized growth factors are also reconstituted usingdionized water and bodily fluids. Suitable bodily fluids forreconstituting freeze-dried growth factors include plasma,hemoconcentrated plasma, whole blood, bone marrow aspirate, andcombinations thereof.

The vacuum product, as above described, contains growth factors with orwithout platelets, leucocytes, or leucocyte-derived cytokines. Thevacuumed product may be used immediately or lyophilized or freeze driedfor future use. In accordance with another embodiment of the presentinvention, the vacuumed product is hemoconcentrated using a 0.2 micronfilter to remove water. Prior to filtering and after the application ofthe sub-atmospheric pressure to the growth factor starting material, thevacuumed product is centrifuged to eliminate from the released growthfactors the cellular components free of growth factors and thecomponents of ghost platelets, bacteria, red blood cells, white bloodcells, and other cellular debris, leaving behind the growth factorcomposition.

A preferred filter is one having a porosity of 0.2 microns or less. Asuitable commercially available filter is the HPH JuniorHemoconcentrator sold by Minntech Therapeutic Technologies. Filteringthe vacuumed and centrifuged product removes water to provide a growthfactor composition of growth factor cytokines and plasma proteins.Centrifuging the vacuumed product separates the extracted growthfactors, plasma proteins and water from the cellular debris, plateletmembranes, ghost platelets, white blood cells, bacteria, and red bloodcells. The centrifugation step is performed as disclosed in the '072patent. It should be understood that the cellular debris cannot becompletely separated from the growth factors without encountering a lossof growth factors.

Growth factors preserved as above described are reconstituted orhydrated in one method using sterile 0.9% normal saline solution. Thepreserved product is also reconstituted using deionized water, sterilewater, other liquid media or bodily fluids including, but not limitedto, plasma, hemoconcentrated plasma, whole blood, bone marrow aspirate,antibiotics or any combination thereof.

In another example of the present invention, 3 milliliters of thepreserved product containing about 70% growth factors is reconstitutedwith about 3 milliliters of 0.9% normal saline or similar liquid media,as discussed above. For wound healing purposes, a therapeuticallyeffective amount of the reconstituted product is applied topically tocover the wound. In another application, it is applied by injection at alocation of soft tissue injury. Beyond wound healing, the fresh productand reconstituted product are useful in medical research applications asbeing supportive of growing tissues or culturing out stem cells. Thereconstituted product may also be a liquid product containingprotein-bound growth factors not previously lyophilized.

The growth factor composition serves as a precursor to the formation ofan extracellular scaffold. The extracellular scaffold is a direct resultfrom the formation of fibrin from fibrinogen. Fibrin is the fibrous,non-globular protein responsible for forming the extracellular scaffold.It is formed by the action of the protease thrombin on fibrinogen whichcauses the latter to polymerize. The polymerized fibrin forms theextracellular scaffold, a fibrin net, or hemostatic plug or clot whichacts as net over a wound site. Factor XIII completes the “cross-linking”of the fibrin strands so that it hardens and contracts. The“cross-linked” fibrin forms a mesh or extracellular scaffold overlyingthe growth factor composition that completes the clotting cascade.

The growth factor composition includes fibrinogen in its current state.The extracellular scaffold is comprised of fibrin in its polymerizedstate. Once the growth factors are released from the platelets, theyreside free in the plasma, creating the growth factor composition. Thegrowth factor composition together with the extracellular scaffold formsthe extracellular tissue matrix. The extracellular scaffold in additionto fibrin includes a collection of extracellular molecules, secreted bycells. The extracellular matrix performs functions, such as cell andtissue adhesion, cell-to-cell communications and differentiation. Theextracellular matrix also provides support, segregates tissues from oneanother and regulates intercellular communication. The extracellularmatrix further functions to regulate a cell's (stem cell) dynamicbehavior. The formation of the extracellular matrix is essential forregenerative processes like growth, wound healing and tissue formationand regeneration.

When the growth factors are released from the platelet granules into theextracellular fluid which is the plasma distinct from the extracellularmatrix, the growth factors are “freed” from the cells but the cellremnants are still in the plasma or growth factor composition. The cellsconstitute the cellular debris, and the growth factors retain theirdistinct beneficial function.

Dependent upon the composition of the growth factor starting material,the released growth factor composition composed of growth factors andcytokines and the associated blood cell remnants or cellular debrisresides in the extracellular fluid or plasma following the applicationof sub-atmospheric pressure. It is not critical to remove the cellulardebris from the growth factor composition. The adhesion moleculesremaining in the cellular debris further contribute to the formation ofthe extracellular scaffold. The extracellular tissue matrix composed ofthe extracellular scaffold and growth factor composition is instrumentalin initiating and supporting the healing and tissue regenerativeprocess.

Prior to preserving the isolated growth factors, in another embodimentvarious pharmaceutical agents are added to the composition. Preferably,these agents are antimicrobial agents which aid in the bioactivity ofwound healing and in the treatment and prevention of infection. Theantimicrobial agents include antibiotics, antiviral and antifungalagents, and the like. However, as known in the art, any number of otherpharmaceutical agents may be employed. The quantity and type of agentselected must be compatible and stable in such products and be capabldof withstanding lyophilization and other methods of preserving thegrowth factor product of the present invention.

In a further embodiment of the present invention, a bodily fluid, suchas blood or an antibiotic, is used to reconstitute the final preservedproduct. This final product allows the clinician a wide berth of optionson how it is used. In another example, by adding bone marrow aspirateand stem cells to the final product the patient achieves the benefitsfrom both therapies. In a similar example, adding liposuction-derivedtissue or surgically obtained adipose derived stem cells to the finalproduct so that the patient achieves the benefits from both of thesetherapies. Additionally, the final product can be administered with anantibiotic solution at a specific anatomical site for wound healing andthe like. Further, when the lyophilized final product and a thrombinsolution are combined, a clot is initiated which is placed in a desiredlocation to promote an effective seal and tissue growth.

The process and growth factor composition of the present invention aresignificant advancements in the field of using platelet based woundtherapy agents. The process allows for the simple preparation of aproduct having one or more growth factors extracted and isolated fromthe growth factor starting material that may be subsequently lyophilizedor preserved for bioactive use at a later date. The growth factorsproduced by the process of the present invention have demonstratedpositive clinical signs in closing wounds and healing damaged softtissue. In this manner large scale production of shelf-ready woundhealing products is facilitated.

The process of the present invention of applying a sub-atmosphericpressure to a volume of growth factor starting material produces agrowth factor composition suitable for a number of uses in woundhealing, therapeutic, surgical, biomedical and cosmetic procedures,tissue regeneration and restoration, and providing bio-scaffoldcomponents and other essential resources to support living cells andtissues. The above applications of the present invention are illustratedin greater detail by the following examples, which should not beconstrued to limit the invention.

EXAMPLE 1

A wound healing composition was prepared aseptically (in a sterilemanner), as diagrammatically illustrated in FIG. 4, by drawing aquantity of whole blood via conventional practice from a patient havinga surface wound. The whole blood was collected in a vial and treatedwith an anticoagulant. At room temperature, the treated blood sample wasplaced in an airtight chamber. A pressure below atmospheric pressure wasapplied to the chamber by a vacuum pump supplied by LabconcoCorporation. The sub-atmospheric pressure released the growth factorsfrom the alpha granules in the platelets to pass through the opencannicular system of the platelets into the extracellular fluid outsidethe platelets. A vial of the extracellular fluid was placed in aconventional blood separation-type centrifuge and spun at about 5,000 to6,000 rpm (“hard spin”) to separate the growth factors, cytokines,plasma proteins and water from red blood cells, other cell remnants andcellular debris. The growth factors, cytokines, plasma proteins andwater were removed from the vial via a syringe and introduced into ahemoconcentrator where the water is filtered out leaving a growth factorcomposition of concentrated growth factors, cytokines and plasmaprotein. The released growth factors were found to contain proteins of aweight in the range of 70-76 kDaltons. The released growth factors wereapplied topically to the area of a surface wound of the patient, and thewound healing was enhanced.

EXAMPLE 2

A wound healing composition was prepared, as diagrammaticallyillustrated in FIG. 5, by drawing a quantity of whole blood from apatient having a soft tissue wound in a tendon in accordance withExample 1 above. The anticoagulated whole blood was then subjected tonegative pressure using a vacuum pump as set forth in Example 1 above toobtain the extracellular fluid containing growth factors. A vial of theextracellular fluid was then centrifuged at about 2,000 to 3,000 rpm,(“soft spin”) to separate the fluid into three layered components, asshown in FIG. 5. The first component included growth factors, cytokines,plasma proteins, and water. The second component included red bloodcells, white blood cells and cell fragments and other debris. The thirdcomponent or bottom layer was composed of packed red blood cells. Thefirst and second components are removed from packed red blood cells andsubjected to a hard centrifugal spin, as described above in Example 1.Thereafter, the product of the hard spin is filtered via ahemoconcentrator, as also described above, to remove the water andprovide a growth factor composition ready for application. The growthfactor composition was then injected into a soft tissue wound of thepatient. The injection of growth factors enhanced healing of the wound.

EXAMPLE 3

A wound healing composition was prepared as diagrammatically illustratedin FIG. 6 from a healthy individual not having a wound by drawing aquantity of whole blood in accordance with Example 1 above. Ananticoagulant was added to the blood. Growth factors were released fromthe whole blood by performing the method steps described in Example 1above. After a hard spin as performed in Example 1, the growth factorcomposition was removed from the layer of packed red blood cells andcellular debris. The unfiltered growth factor composition was thendivided into a plurality of individual equal volumes, such as vials of 3ml. liquid samples shown in FIG. 6. Each square shown in FIG. 6represents an individual vial of growth factor composition ready for useor storage. The collection of samples was lyophilized in a conventionalmanner to provide a preserved, shelf-stable product. After a preselectedperiod of time after lyophilization, two sample vials of preservedgrowth factors were reconstituted or hydrated by mixing the preservedgrowth factors with a sterile 0.9% normal saline solution. One quantityof the reconstituted growth factors was applied topically to the area ofa surface wound of one patient. The second quantity of the reconstitutedgrowth factors was injected into a soft tissue wound of a secondpatient. In each patient the wound healing took place in an enhancedmanner.

The wound healing compositions prepared in accordance with the presentinvention are not limited to the uses disclosed in Examples 1-3 above.Filtered and unfiltered growth factors prepared by the process of thepresent invention have many other uses. Some additional examples (notall inclusive) include the treatment of injuries to tendons andligaments, bone and joint healing, oral and maxillofacial surgery, hairgeneration and stem cell recruitment, skin rejuvenation, and applicationwith stem cells to create a three dimensional organ bio-scaffold to beseeded with progenitor cells.

The application of a sub-atmospheric pressure to tissues is not limitedto growth factor starting materials. The application of sub-atmosphericpressure has been demonstrated to significantly reduce or eliminate thenormal cellular structure of processed whole blood and blood cells. Theprocess can be applied to animal tissues composed of cells to eliminateintact cell structures throughout, leaving behind an acellular tissuematrix. The tissue matrix is washed with one or more sterilebiocompatible solution(s) to remove devitalized, diseased or pathologiccompromised tissue cells, bacteria, undesirable cellular debris orcomponents retaining antigenicity. Clearing of such tissue of itscellular components facilitates application of a growth factorcomposition described herein along with stem cell reseeding of theacellular tissue matrix in an in vitro environment supportive of stemcell activity. Such a process using the products produced by theapplication of sub-atmospheric pressure promotes autologous, allogeneic,or xenogeneic sourced organ regeneration for potential transplantation.

By using a selected one of the methods described in Examples 1, 2, or 3above, applications of growth factor compositions are applied byinjection, spray infusion or topically where appropriate. The growthfactor compositions are applied to traumatized tissues of human, canineand or equine subjects affected with muscle-skeletal injuries and jointconditions, autoimmune disorders, respiratory tract conditions, ocularconditions, neurologic trauma and disease related pathology. Treatmentof dermal thermal burns and dental related procedures are enhanced andaccelerated by application of growth factor compositions prepared inaccordance with the present invention. The growth factor composition isalso used in the treatment of diseased tissue including immune mediateddiseases, such as alopecia areata, inflammatory diseases such asmultiple sclerosis and COPD, degenerative diseases and procedures usedin hair reproduction and organ growth for transplantation.

Due to multiple factors, including age and diurnal fluctuations inplatelet counts, benefits in individual tissue responses vary. Althoughvery unlikely, risks associated with autologous use of growth factorcompositions are limited to contaminants or additional trauma introducedduring the application process. Allogeneic or xenogeneic growth factorcompositions may additionally result in protein related allergenicreactions due to individual patient sensitivity. The transfer of certainmicroorganisms, such as a virus, can be potentially eliminated byprescreening of donors.

According to the provisions of the patent statutes, we have explainedthe principle, preferred construction and mode of operation of ourinvention and have illustrated and described what we now consider torepresent its best embodiments. However, it should be understood thatwithin the scope of the appended claims the invention may be practicedotherwise than as specifically illustrated and described.

We claim:
 1. A process for obtaining growth factors comprising the stepsof: providing a preselected volume of unfrozen growth factor startingmaterial wherein the unfrozen growth factor starting material containingplatelets is obtained from the tissue of a subject, applying apreselected sub-atmospheric pressure to the unfrozen growth factorstarting material to extract growth factors from the platelet granulesof the unfrozen growth factor starting material without activating theclotting process within the growth factor starting material, releasingthe extracted growth factors from the cellular structure of the growthfactor starting material into extracellular fluid to provide anextracellular growth factor composition containing the platelet granulegrowth factors, collecting the extracted growth factors for mixture witha nondestructive medium without dehydrating the growth factor startingmaterial, and containing in the nondestructive medium a therapeuticallyeffective amount of growth factors for creating a positive reaction onliving tissue.
 2. A process as set forth in claim 1 which includes,extracting the growth factors by the application of sub-atmosphericpressure from within the platelet granules through the platelet outercell membrane into the growth factor composition external of theplatelet.
 3. A process as set forth in claim 1 which includes,eliminating from the released growth factors the cellular componentsincluding alpha and dense granules free of growth factors and thecomponents of ghost platelets, white blood cells, red blood cells, andother cellular debris contained in the growth factor starting materialleaving behind the growth factor composition.
 4. A process as set forthin claim 3 which includes, using the growth factor composition resultingfrom the release of the growth factors from the growth factor startingmaterial to form an extracellular tissue matrix supportive of stem cellreseeding in an in vitro environment.
 5. A process as set forth in claim1 which includes, reducing the cellular debris content from the growthfactors as the growth factors are extracted from the growth factorstarting material for the formation of a growth factor composition, andusing the growth factor composition as a framework for creating a tissuematrix for use as a foundation for regenerative processes.
 6. A processas set forth in claim 1 which includes, subjecting the growth factorstarting material to a preselected sub-atmospheric pressure for apreselected period of time to extract growth factors from the growthfactors starting material.
 7. A process as set forth in claim 6 whichincludes, applying sub-atmospheric pressure to the growth factorstarting material at a pressure inversely proportional to the period oftime the sub-atmospheric pressure is applied to the growth factorstarting material.
 8. A process as set forth in claim 1 which includes,applying the sub-atmospheric pressure to the growth factor startingmaterial in a range between about 5 millibars to 1 atmosphere and at atemperature between about 1° C. to 37° C.
 9. A process as set forth inclaim 1 which includes, accumulating the released growth factors in asurrounding nondestructive medium in a bioactive state to promote tissuegrowth.
 10. A process as set forth in claim 1 which includes, separatingthe extracted growth factors from the confines of the alpha and densegranule membranes of the platelets.
 11. A process as set forth in claim1 which includes, separating the extracted growth factors having amolecular weight in the range 70-76 kDaltons from the cellular structureof the growth factor starting material.
 12. A process as set forth inclaim 1 which includes, preserving the extracted growth factors forselected bioactive use by preservation methods selected from a groupconsisting essentially of lyophilization, cryopreservation, and flashdrying.
 13. A process as set forth in claim 12 which includes,reconstituting lyophilized solution growth factors with a sterile 0.9%normal saline.
 14. A process as set forth in claim 12 which includes,reconstituting lyophilized growth factors using a selected one ofdeionized water, sterile water, and bodily fluids.
 15. A process as setforth in claim 14 which includes, using a bodily fluid selected from thegroup consisting essentially of plasma, hemoconcentrated plasma, wholeblood, bone marrow aspirate, and combinations thereof to reconstitutefreeze-dried growth factors.
 16. A process as set forth in claim 13which includes, topically applying a therapeutically effective amount ofreconstituted growth factors selectively to internally and externallydamaged tissue sites to promote healing thereof.
 17. A process as setforth in claim 1 which includes, filtering the released growth factorsusing a 0.2 micron filter to remove water from the growth factorcomposition.
 18. A process as set forth in claim 1 which includes,preventing activation of plasma when growth factors are released fromthe growth factor starting material by employing sub-atmosphericpressure to separate the growth factors from the growth factor startingmaterial, combining the released growth factors with the plasma to forma growth factor composition including fibrinogen, converting fibrinogenin the growth factor composition to fibrin to form an extracellularscaffold, and combining the growth factor composition with theextracellular scaffold to form an extracellular tissue matrix forcreating a positive reaction on living tissue.
 19. A process as setforth in claim 1 which includes, controlling the time for extractinggrowth factors from the growth factor starting material.
 20. A processfor producing a therapeutically and regeneratively effectivebiofunctional agent comprising the steps of: applying a preselectedsub-atmospheric pressure to growth factor starting material to extractgrowth factors from platelets of the growth factor starting materialderived from the tissue of a subject without activating the growthfactors and with the growth factors remaining in a natural state,producing a growth factor composition by combining growth factorsextracted from platelets with plasma, converting fibrinogen in thegrowth factor composition to fibrin to form an extracellular scaffold,and combining the growth factor composition with the extracellularscaffold to form an extracellular tissue matrix for creating a positivereaction on living tissue.
 21. A process as set forth in claim 20 whichincludes, washing the extracellular tissue matrix with a sterilebiocompatible solution to remove therefrom devitalized, diseased orotherwise compromising tissue cells, bacteria, and other undesirabledebris.
 22. A process as set forth in claim 20 which includes, applyingthe extracellular tissue matrix with stem cells in an in vitroenvironment supportive of stem cell activity.
 23. A process as set forthin claim 22 which includes, promoting cell growth and differentiationinto specific tissue cell types within stem cell seeded cells mixed withthe tissue matrix substantially free of cellular debris in the nature oftissue cells, platelets, ghost platelets, which blood cells, red bloodcells, and bacteria.
 24. A process as set forth in claim 22 whichincludes, combining the extracellular tissue matrix with stem cells topromote tissue growth.
 25. A process for producing a therapeutically andregeneratively effective biofunctional agent comprising the steps of:extracting growth factors from platelets of growth factor startingmaterial derived from the tissue of a subject without activating thegrowth factors and the growth factors remaining in a natural state,producing a growth factor composition including growth factors extractedfrom platelets in plasma and extracellular fluid, separating the growthfactor composition from the growth factor starting material, collectingthe growth factor composition, and applying the growth factorcomposition in the treatment of diseased tissue including immunemediated diseases including alopecia areata, inflammatory diseasesincluding multiple sclerosis and COPD, degenerative diseases andprocedures used in hair reproduction and organ growth fortransplantation.